Immuno-chemical Assays
Several different substrates are known in the part to be used for enzyme systems in connection with peroxidase-based immuno-chemical assays, for example ELISA. These substrates are often toxic, mutagenic or carcinogenic.
ELISA (Enzyme Linked Immuno Sorbent Assay) is a method used to assess the amount of antibody in serum. The main principle is that in a much diluted solution many antigens will become bound to the surface of plastic. Thus, if a much diluted solution of antigens is incubated for a time in plastic trays it is possible to wash the cavities in a buffer solution and still retain the film of antigens on the surface of the plastic. If it is required to determine the amount of antibodies in, for instance, serum then the trays with their deposits of antigens are incubated with the serum, The antibodies attach themselves to the antigens and, after thorough washing the trays are again incubated this time with a marker, for example anti-immune globulin serum to which there is attached a suitable enzyme by covalent bonding. In this particular case peroxidase is used.
The peroxidase-marker complex will attach itself to those locations where there is already a deposit of antibodies. After thorough washing to remove all un-combined material the enzyme activity is measured, normally by the use of a suitable colour indicator. Enzyme activity may be determined by the addition of a cromogenetic substrate (i.e. colour producing compound) and hydrogen peroxide. The enzyme catalyses the reduction of the substrate to a coloured compound and the resultant degree of absorbency provides a measure of the enzyme activity. If the serum contains no antibodies there will be no enzyme activity, on the other hand if there is much antibody present there will be very considerable enzyme activity. A standard curve can be drawn showing enzyme activity as a function of the concentration of antibodies. This may be used to estimate the content of antibodies in unknown serum samples by interpolation.
Many of the peroxidase substrates are aromatic amines and include diaminobenziden (DAB), 3,3'-diamino benzid tetra hydrochloride, 3,3',5,5'-tetra methyl benzidin (TMD). Another peroxidase substrate, which does not belong to the group of aromatic amines, is 2,2-azino-di (3-ethyl-benzo thiazolin-6-sulphonic acid) (AEBTS), this has been used as a standard for the establishment of the activity of peroxidase preparations. According to Voogd, Van der Stel and Jacobs (1980) this material is also a mutagent.
o-phenylendiamine (OPD) is another peroxidase substrate which is widely used in hospital and development laboratories. OPD is known to be both mutagenic and carcinogenic.
The staff of laboratories in which analyses involving the use of toxic, mutagenic or carcinogenic materials are carried out are exposed to a significant degree of risk of coming into direct contact with these materials. In order to provide a safe working environment considerable efforts are now made to substitute these dangerous materials with less dangerous ones.
Hair Dyeing Composition
In addition to being used as a substrate in immuno-chemical assays OPD is used to dye hair. In this connection too it is desirable to substitute a dangerous material with one less dangerous so that the user is not exposed to danger by coming into contact with it. To protect the hands against the dangerous material it is normal for gloves to be used while the hair dye is being applied. Gloves cannot, of course, protect the scalp of the person to whom the dye is applied.
In general hair dyeing compositions on the market today can be divided into three main groups:
temporary hair dyes, PA1 semi-permanent hair dyes, and PA1 permanent oxidative hair dyes. PA1 R is an amino, mono- or a distributed amino or OR', where R is H, alkyl, alkenyl, alkynyl, halogenalkyl, nitro, benzyl, phenyl or substituted phenyl. X, Y and Z may each be any one of the following: alkyl, alkenyl, alkynyl, halogenalkyl, nitro, benzyl, phenyl, substituted phenyl, amino, hydroxy or mercapto with the proviso that at least one of the groups X, Y and Z is an amino group or an amino salt. PA1 a) Laccases or related enzymes, which act on molecular oxygen and yield water (H.sub.2 O) without any need for peroxide (e.g. H.sub.2 O.sub.2), PA1 b) Oxidases, which act on molecular oxygen (O.sub.2) and yield peroxide (H.sub.2.sub.2 O), and PA1 c) Peroxidases, which act on peroxide (e.g. H.sub.2 O.sub.2) and yield water (H.sub.2 O)
The temporary hair dyes are only intended to change the natural hair colour for a short period of time and usually functions by depositing dyes on the surface of the hair. Such hair dyes are easy to remove with normal shampooing.
When using semi-permanent hair dyes the colour of the dyed hair can survive for five or more shampooings. This is achieved by using dyes having a high affinity for hair keratin and which is able penetrate into the interior of the hair shaft.
Permanent hair dyes are very durable to sunlight, shampooing and other hair treatments and need only to be refreshed once a month as new hair grows out. with these dyeing systems the dyes are created directly in and on the hair. Small aromatic colourless dye precursors (e.g. p-phenylene-diamine, o-aminophenol, o-phenylendiamine (OPD)) penetrate deep into the hair where said dye precursors are oxidised by an oxidising agent into coloured polymeric compounds. These coloured compounds are larger than the dye precursors and can not be washed out of the hair.
By including compounds referred to as modifiers (or couplers) in the hair dyeing composition a number of hair colour tints can be obtained. Cathecol and Resorcinol are examples of such modifiers.
Some of the today most widely used dye precursors such as OPD are known to be both mutagenic and carcinogenic.
Further, traditionally H.sub.2 O.sub.2 is used as the oxidizing agent (colour builder), but also as a bleaching agent. Dyeing compositions comprising H.sub.2 O.sub.2 are often referred to as "lightening dyes" due to this lightening effect of H.sub.2 O.sub.2.
The use of H.sub.2 O.sub.2 in dyeing compositions have some disadvantages as H.sub.2 O.sub.2 damages the hair. Further, oxidative dyeing often demands high pH (normally around pH 9-10), which also inflicts damage on the hair and on the skin. Consequently, if using dye compositions comprising H.sub.2 O.sub.2 it is not recommendable to dye the hair often.
To overcome the disadvantages of using H.sub.2 O.sub.2 it has been suggested to use oxidation enzymes to replace H.sub.2 O.sub.2.
U.S. Pat. No. 3,251,742 (Revlon) describes a method for dyeing human hair by dye formation in situ (i.e. on the hair). An oxidation enzyme is used for the colour formation reactions at a substantially neutral pH (7-8.5). Laccases, tyrosinases, polyphenolases and catacolases are mentioned as suitable oxidation enzymes. The hair colour pigment is formed by controlled oxidation of various quinone-forming compounds and mono or poly aromatic amines having the amino groups on the aromatic rings to form natural appearing pigments. Specifically mentioned dye precursors are 2-amino-4-nitrophenol, p-phenylene diamine, m-phenylene diamine, o-phenylene diamine, 2-amino-1,4-naphthoquenone, m-aminophenol, p-aminophenol, o-aminophenol, 2-amino resorcinol, 1,2,4-benzene triamine, nitro-p-phenylene diamine, 2-amino-5-diethyl amino toluene.
EP patent no. 504.005 (Perma S. A.) concerns dyeing compositions for keratinous fibres, in particular hair, which do not require the presence of H.sub.2 O.sub.2 (hydrogen peroxide). The composition comprises an enzyme capable of catalysing the formation of the polymeric dyes and also dye precursors, such as bases and couplers, in a buffer solution wherein the pH of said composition is between 6.5 and 8 and said enzyme has an optimal activity in the pH range between 6.5 and 8. Rhizoctonia praticola laccase and Rhus vernicifera laccase are exemplified as the oxidation enzyme to oxidize the dye precursor(s). The following dye precursors are specifically mentioned: p-phenylene diamine, o-aminophenol, p-methylaminophenol, p-aminophenol, p-toluylenediamine and N-phenyl-p-phenylene diamine.
The aim of the present invention is to use the present findings to make available a substrate which to all intents and purposes is non-toxic, non-mutagenic and/or non-carcinogenic and which may be used in immuno-chemical assays, for the dying of keratinous fibres, in particular hair and for dying both natural and synthetic fibres, e.g. textiles. This aim is achieved by utilising the discovery of a substrate which includes the group with the general formulae shown in 1. ##STR1##
wherein
In a special embodiment of the invention a substrate is made available which includes a connection with formula 1 where R' is a methyl, ethyl or iso propyl group.
In an preferred embodiment the substrate is a benzoic acid ester, in particular 3,4-diaminobenzoic acid methyl ester (DABA-Me), 3,4-diaminibenzole acid ethyl ester and 3,4-diamino benzoic acid isopropyl ester.
Comparison of the very toxic aniline with the carboxyl acid derivative of aniline, p-amino benzoic acid (PABA) shows that the toxic city of the molecule is radically altered by the addition of the carboxyl group. PABA is generally considered to be non-toxic and, among other applications, is used as an ultra violet filter in sun lotions. If the substrate OPD is thought of along the same lines it can be seen that a possible analogue is 3,4-diamino benzoic acid (3,4-DABA) n This material is comparatively cheap and readily obtainable. ##STR2##
Investigation using enzymes showed that 3,4-DABA is a considerably poorer substrate for peroxidase than is OPD. In other words 3,4-DABA has a higher K.sub.m -value at the same V.sub.max when compared with OPD. This is apparently due to the carboxyl group's inductive (deactivating) effect upon the aromatic ring. This effect can be countered by modifying the carboxyl group, for example by esterification with an alcohol. The preferred alcohols are methanol, ethanol and iso propcanol. Methyl, ethyl and isopropyl esters were examined in connection with enzymes and the materials were shown to have significantly improved properties than 3,4-DABA. Especially the ethyl ester was found to have a very high V.sub.max, i.e. at the same concentration it gives a very much higher reaction speed than OPD.
The reaction mechanism for the oxidation of OPD with, for instance, hydrogen peroxide, both with and without an enzyme, is described by the following equations: ##STR3##
It may be seen from this that the product of oxidation is 2,3-diamino phenazin.
When one of the compounds described here is employed the product of oxidation is also an amino phenazine. In the case of 3,4-DABA the oxidation product is 4,7-dicarboxy-1,2-diamino phenazine as shown in the following equation. ##STR4##
The substrate should have two amino groups at the 3,4-location for the reaction to take place. However, substrates which have the amino groups at either the 2,3-location or the 3,4-location may be used but only if the carboxyl group does not hinder the reaction.
Substrates which have the amino group at the para location may also be used to produce a coloured product. An example of this is 3,6-DABA.
In order to counteract the carboxyl groups inductive effect (i.e. deactivation of the aromatic ring caused by the groups attractive effect upon electrons) esterification of the carboxyl group was investigated using electron donating groups to see if deactivation could be counteracted while at the same time retaining non-mutagenic attributes. To investigate the effect of different alkyl groups on the material's enzymatic properties as well as possible mutagenic properties the methyl, ethyl and isopropyl esters of 3,4-DABA were synthesised.
Compounds with the general formulae 1 are preferably dissolved in DMF (Dimethyl formamide) but other organic solvents may be used for this purpose. If a compound with the general formulae 1 is in the form of a salt it can be dissolved in water and this is preferred when using organic solvents.
In another aspect the invention relates to a method for quantitative and/or qualitative analysis of a material of biological interest. In this case a peroxidase enzyme together with a marker is bound to the compound in question. Hydrogen peroxide is then converted with a cromogenetic substrate (i.e. colour forming compound) in the presence of the peroxidase, the substrate includes a bond with the general formulae 1.
In a preferred embodiment of the method of the invention one of the following substrates are used: the methyl, propyl or isopropyl ester of amino benzoic acid.
In those cases where the material of biological interest is an antigen the associated antibody is used. In this connection other combinations will suggest themselves to the skilled person.
The coloured product produced by the method of the invention is especially suited to the dying of textiles, thread, yarn, wool, hides and skins and human hair. Other natural fibres such as cotton and silk may also be dyed with the product as may synthetic fibres such as polyamides, polyurethane and polyester.
The coloured product may either be made immediately before it is to be used for dying or it may be synthesised in the immediate vicinity of the substance to be dyed. For example this may be done by mixing the substrate and the oxidation system in a person's hair.
The dyeing process may be carried out rinsing the person's hair with a mixture of the substrate of the invention and hydrogen peroxide or an oxidation enzyme. A peroxidase is then added and distributed in the hair. When the desired degree of colouring has been obtained the hair is rinsed with water.
The substrate may be mixed with the oxidation system before it is applied to the hair. As stated above the substrate may be oxidised with hydrogen peroxide or an oxidation enzyme generating hydrogen peroxidase in the presence of a peroxidase.
Peroxidases belongs to the group of enzymes which is known as the oxidoreductases. The group also includes the classes of enzymes dehydrogenase, oxygenase, oxidase, laccase and related enzymes. These enzymes may also be used for as an oxidation system/agent for e.g. dyeing keratinous fibres, such as hair, wool, fur and hides and the like. Dyeing composition and preferred oxidation enzymes will be described further below.
In oxidation reactions which are catalysed by the enzyme peroxidase the oxygen donor is hydrogen peroxide which is used as an electron acceptor. Oxidases employ oxygen as an electron acceptor.
Examples of suitable oxidases include catecholoxidase, laccase and o-amino phenoloxidase.
Oxidation systems which may be used for the oxidation of the substrate in this connection therefore include peroxidase and hydrogen peroxide as well as oxidases, laccases and related enzymes and oxygen. When the system consists of only an oxidase and oxygen it is only necessary to add the oxidase to the substrate as the oxygen in the air is used as an oxidant.
Dyeing Composition
In an aspect the invention relates to a composition in particular adapted for dyeing keratinous fibres comprises, e.g. hair, fur, hide or wool. Comprising 1) at least one oxidation enzyme 2) at least one substrate as defined by the formulae 1 and optionally 3) at least one modifier.
A preferred use of the composition is as a permanent dye for the dyeing of human hair.
The oxidation enzyme is as also indicated above an oxidoreductase, i.e. an enzyme classified under the Enzyme Classification number E.C. 1 (Oxidoreductases) in accordance with the Recommendations (1992) of the International Union of Biochemistry and Molecular Biology (IUBMB)) which catalyses oxidoreduction reactions.
Within the class of oxidoreductase enzymes are preferred enzymes which catalyse the oxidation of a substrate (an electron or hydrogen donor) by acting on oxygen (O.sub.2) and/or a peroxide as the acceptor. Such enzymes include enzymes classified within the enzyme classes comprising oxidases, including E.C. 1.1.3. E.C. 1.2.3, E.C. 1.3.3, E.C. 1.4.3, E.C. 1.5.3, E.C. 1.7.3, E.C. 1.8.3 and E.C. 1.9.3, laccases and related enzymes in E.C. 1.10.3, and peroxidases in E.C. 1.11.
According to the invention three types of oxidoreductases are specifically contemplated:
Also, enzyme systems which comprise a combination of more than one enzyme from a single class or from different classes among the three types of enzymes are contemplated. In the present specification, although reference will often be made to a single enzyme for the sake of simplicity, it is to be understood that the description is generally applicable to such combinations of more than one enzyme. Further, although the invention is generally described in terms of the preferred aspect relating to the dyeing of hair, it is to be understood that the description is generally applicable to compositions according to the invention adapted for dyeing of other types of keratinous fibres.
Particularly preferred enzymes are laccases and related enzymes, the term "laccases and related enzymes" including enzymes comprised by the enzyme classification E.C. 1.10.3.2 (laccases) and catechol oxidase enzymes comprised by E.C. 1.10.3.1, bilirubin oxidase enzymes comprised by the enzyme classification E.C. 1.3.3.5 and mono-phenol mono-oxygenase enzymes comprised by the enzyme classification E.C. 1.14.99.1. Laccases are multi-copper containing enzymes that catalyze the oxidation of phenols and aromatic amines. Laccase-mediated oxidation results in the production of aryloxy-radical intermediates from suitable phenolic substrates; the ultimate coupling of the intermediates so produced provides a combination of dimeric, oligomeric, and polymeric reaction products. Certain reaction products can be used to form dyes suitable for dyeing hair.
Preferably, the laccase employed may be derived from a strain of Polyporus sp., in particular a strain of P. pinsitus or P. versicolor, a strain of Myceliophthora sp., e.g. M. thermophila, a strain of Rhizoctonia sp., in particular a strain of Rh. praticola or Rh. solani, a strain of a Rhus sp., in particular Rhus vernicifera, a strain of Pyricularia sp, in particular P. oryzae, or a strain of Scytalidium, such as S. thermophilium.
In specific embodiments of the invention the oxidoreductase is a laccase such as a Polyporus sp. laccase, especially the Polyporus pinisitus laccase (also called Trametes villosa laccase) described in WO 96/00290 (from Novo Nordisk Biotec Inc.) or a Myceliophthora sp. laccase, especially the Myceliophthora thermophila laccase described in WO 95/33836 (from Novo Nordisk Biotech Inc.).
Further, the laccase may be a Scytalidium sp. laccase such as the S. thermophilium laccase described in WO 95/33837 and WO 97/19998 (from Novo Nordisk Biotech Inc.), the contents of which is incorporated herein by reference, or a Pyricularia sp. laccase, such as the Pyricularia oryzae laccase which can be purchased from SIGMA under the trade name SIGMA No. L5510, or a Coprinus sp. laccase, such as a C. cinereus laccase, especially a C. cinereus IFO 30116 laccase, or a Rhizoctonia sp. laccase, such as a Rh. solani laccase, especially the neutral Rh. solani laccase described WO 95/07988 (from Novo Nordisk A/S) having a pH optimum in the range from 6.0 to 8.5.
The laccase may also be derived from a fungus such as Collybia, Fomes, Lentinus, Pleurotus, Aspergillus, Neurospora, Podospora, Phlebia, e.g. P. radiata (WO 92/01046), Coriolus sp., e.g. C. hirsitus (JP 2-238885), or Botrytis.
Bilirubin oxidase may preferably be derived from a strain of Myrothecium sp., such as M. verrucaria.
The substrates (i.e. dye precursors) may according to the dyeing composition of the invention be any of the above within the definition of the general formulae 1.
Preferred dye precursors (i.e. substrates) are benzoic acid esters, especially diamino benzoic acid esters, in particular 3,4-diamino benzoic acid methyl ester (DABA-Me), 3,4-diamino benzoic acid ethyl ester and 3,4-diamino benzoic acid isopropyl ester.
Other Oxidation Agents
The substrate may also be oxidised by a number of inorganic compounds, among these are compounds which include hypochlorite (ClO.sup.-), hypobromite (BrO.sup.-), permanganate (MnO.sub.4.sup.-) dicromate (Cr.sub.2 O.sub.7.sup.2-) and the iron ion (Fe.sup.3+).
Oxidation systems are taken to be either an oxidant per se or a combination of an enzyme and an oxidant.
Modifiers
Modifiers typically incorporation in a dye compositions include m-aromatic diamines, m-aminophenols, polyphenols, amino naphthalines or naphthols. The modifier (coupler) reacts with the dye precursor in the presence of the oxidative enzyme or the like, converting it into a coloured compound. Examples of specific modifiers (couplers) include m-phenylene-diamine, 2,4-diaminoanisole, 1-hydroxynaphthalene (.alpha.-naphthol), 1,4-dihydroxybenzene(hydroquinone), 1,5-dihydroxynapthalene, 1,2-dihydroxybenzene(pyrocatechol), 1,3-dihydroxybenzene (resorcinol), 1,3-dihydroxy-2-methylbenzene, 1,3-dihydroxy-4-chlorobenzene (4-chlororesorcinol), 1,2,3, trihydroxybenzene, 1,2,4-trihydroxybenzene, 1,2,4-trihydroxy-5-methylbenzene, and 1,2,4-trihydroxytoluene.
Method of Dyeing Keratinous Fibres
In a further aspect the invention relates to a method for dyeing keratinous fibres, in particular hair, fur, hide and wool, using a composition as described above. The dyeing method can be conducted with one or more dye precursors (i.e. substrates of the invention) and optionally in combination with one or more modifiers. The amount of dye precursor(s) and other ingredients used in the composition of the invention for this purpose are in accordance with usual commercial amounts and therefore known for the skilled person. Hair dyeing is typically carried out at or near room temperature, preferably around the optimum temperature of the enzyme being used, and at a pH in the range of from 3.0 to 9.0, preferably 4.0 to 8.5, especially 6.0 to 8.0. Dye precursors (i.e. substrates of the invention) and optional modifiers are described above.